In the proposed research we plan to extend our investigations of the relation between structure and function in proteins. Spatial and temporal fluctuations appear to be essential ingredients in biomolecular action. Information on such fluctuations can be obtained in a variety of ways. By using flash photolysis over extended ranges in time (ps to ks), temperature (4-300 K), and pressure (0.1-600 MPa) on a wide variety of systems (heme proteins, bacteriorhodopsin) we will obtain detailed information on the multiple steps involved in binding and catalytic processes, on the distribution of conformational substates, and on conformational relaxation. By investigating the shapes of optical and Raman lines as functions of pressure, temperature, and time, we expect to learn more about the conformational substates and their temporal behavior. Quantum-mechanical tunneling below about 100 K will provide data on the width of the innermost barrier in heme proteins. To study the spatial aspects, we will, in collaboration with othe groups, use two techniques. X-ray diffraction over the temperature range from about 50-300 K yields insight into spatial fluctuations of each atom and, in particular, the shape of the potential in which each atom moves. EXAFS experiments at low temperature, combined with photodissociation, give additional information on the structural changes occurring at the active center during ligand binding. We believe that the total information that we hope to gather from the wide range of techniques will be much to elucidate the dynamic aspects of protein function.